標題: 三帖皂?生合成酵素之蛋白質工程及其產生具生物活性化合物之研究
The Research on Protein Engineering of Triterpene Saponin Biosynthetic Enzymes for Bioactive Compounds Production
作者: 吳東昆
WU TUNG-KUNG
國立交通大學生物科技學系(所)
關鍵字: 氧化鯊烯環化酵素;三萜皂苷;三萜類生合成修飾酵素;氫氧化酵素;醣基轉移 酵素;oxidosqualene cyclase;triterpene saponin;triterpene tailoring enzyme;hydroxylase;glycosyltransferase
公開日期: 2014
摘要: 三萜皂苷生合成酵素之蛋白質工程及其產生具生物活性化合物之研究 三萜皂苷化合物由一系列具親脂性的三萜類單元以及親水性的醣基單元所組成。三萜 皂苷化合物普遍存在於植物、黴菌、與海洋生物。最近之研究顯示三萜皂苷化合物具有包 括抗發炎、抗黴菌、抗細菌、抗寄生蟲、及抗腫瘤等多樣之生理活性。由三萜皂苷化合物 之生合成研究,顯示其結構多樣性主要是由:(1)氧化鯊烯環化及重組成不同之多個立體 中心之多環產物;(2)在環化產物之多個不同之碳原子之位置進行氫氧基化或氧化;(3)在 氫氧基位置進行醣基轉移反應形成最終之皂苷產物。在本實驗室之前的研究中,我們利用 基因工程結合產物分離與鑑定之技術,探討酵母菌之氧化鯊烯環化酵素所催化之環化重組 反應之結構-功能-作用機制關係,並由其中分離出多個在環化或重組過程中被中斷之中間 產物。這些結果支持酵素之結構-功能-作用機制關係以及提供作為未來藥物應用之基礎。 另外,在所產生之中間產物,利用三萜類生合成之修飾酵素進行官能基或衍生物之修飾, 將可增加其結構之多樣性以及幫助其生理活性。 為了進一步探討氧化鯊烯環化酵素所催化之環化重組反應之結構-功能-作用機制關係, 並獲得更多具結構多樣性之三萜類化合物,我們將繼續對酵母菌及其它物種之氧化鯊烯環 化酵素進行蛋白質工程,以期能獲得更多具結構多樣性之中間或非天然產物。同時我們也 將選殖及表現三萜類生合成途徑之修飾酵素如氫氧化酵素及醣基轉移酵素進行in vitro及 in vivo之蛋白質表現。經in vitro表現之蛋白質將先進行純化及原先受質之活性測試。同 時,上述蛋白亦將進行in vivo之共表現以使其直接生成三萜皂苷化合物。接著,則將針 對所生合成出之中間或非天然產物之三萜皂苷化合物進行溶血、抗菌及抗腫瘤藥物之活性 測試。本計畫在未來三年之工作目標將進行以下數項之工作: (1)不同物種之氧化鯊烯環化酵素之基因合成及分子選殖和蛋白質表現。 (2)不同物種之氧化鯊烯環化酵素之蛋白質工程生合成裁製修飾截短或神奇之產物。 (3)不同物種之氧化鯊烯環化酵素之變種產生之修飾截短或神奇產物之分離與結構確 認。 (4)三萜類生合成途徑之氫氧化修飾酵素之基因序列最適化、合成、分子選殖、功能性 表現、及蛋白質純化。 (5)三萜類生合成途徑之醣基轉移酵素之基因序列最適化、合成、分子選殖、功能性表 現、及蛋白質純化。 (6)三萜類生合成途徑之氫氧化修飾酵素及醣基轉移酵素對於其原生受質之活性分 析。 (7)三萜類生合成途徑之修飾酵素之蛋白質工程及對於其原生受質之活性分析。 (8)三萜皂苷衍生化合物於溶血活性之活性分析。 (9)三萜皂苷衍生化合物於抗腫瘤活性之活性分析。 (10)三萜皂苷衍生化合物於抗菌活性之活性分析。
The Research on Protein Engineering of Triterpene Saponin Biosynthetic Enzymes for Bioactive Compounds Production Triterpene saponins constitute a family of compounds with a hydrophobic triterpene aglycone and hydrophilic sugars structures. They are widely distributed in various natural sources including plants, fungi, and marine organisms. Recent researches showed that the triterpene saponins exhibit diverse biological properties including antiinflammatory, antifungal, antimicrobial, antiparasitic, and antitumor activities. Biosynthetic studies suggested that the triterpene saponin structure diversity is generated by (1) cyclization of oxidosqualene to various triterpene skeletons, (2) hydroxylation or oxidation at multiple positions, and (3) glycosylation to add sugar moieties through an ether or ester glycoside linkage. We have applied genetic engineering coupled with product characterization to elucidate the structure-function-reaction mechanism relationships of the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase-catalyzed (ERG7) cyclization/rearrangement reaction. Numerous truncated or altered deprotonation products were isolated from the ERG7 mutants. Modification of triterpene-type core structures with tailored enzymes holds great potential both in increasing structure diversity and in enhancing biological activity. To further explore the structure-function-reaction mechanism relationships of oxidosqualene cyclases and to obtain triterpenes with more structural diversity, we continue in this proposal to engineer SceERG7 and related oxidosqualene cyclase genes from various species to create more novel truncated or unnatural triterpene structures. In parallel, genes of triterpene tailoring enzymes such as hydroxylases and glycosyltransferases will also be obtained for in vitro or in vivo expression. The in vitro expressed triterpene tailoring enzymes will be purified and used to modify the isolated triterpene compounds. Alternatively, the novel oxidosqualene cyclase and the triterpene tailoring enzyme genes will be cloned and in vivo co-expressed to direct synthesize tailored triterpene products. The biological functions ofthe produced tailored triterpene saponins will be evaluated for their antimicrobial, antifungal, and antitumor activity. For the period of the proposal, several specific goals will be achieved as listed below: (1) Various artificial OSC genes synthesis and molecular cloning for the proteins expression. (2) Protein engineering of various oxidosqualene cyclases for novel truncated or unnatural triterpene biosynthesis. (3) Isolation and characterization of novel truncated or unnatural triterpene products produced by OSC mutants. (4) Sequence optimization, synthesis, molecular cloning, functional expression, and protein purification of triterpene hydroxylases. (5) Sequence optimization, synthesis, molecular cloning, functional expression, and protein purification of triterpene glycosyltransferase. (6) Activity assay of triterpene hydroxylases and glycosyltransferases on their native substrates. (7) Protein engineering of triterpene tailoring enzymes on novel truncated or unnatural triterpene products isolated from OSC mutants. (8) Biological assay of triterpene saponin derivatives on hemolytic activity. (9) Biological assay of triterpene saponin derivatives on antitumor activity. (10) Biological assay of triterpene saponin derivatives on antimicrobial activity.
官方說明文件#: NSC102-2113-M009-017-MY3
URI: http://hdl.handle.net/11536/97161
https://www.grb.gov.tw/search/planDetail?id=8110942&docId=429772
Appears in Collections:Research Plans